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Abstract

Neovascularization is an important feature in advanced atherosclerosis. It is recognized by the formation of new blood vessels recruited from the adventitia that penetrate the plaque, presumably due to lack of oxygen (hypoxia) in the plaque core. These immature, leaky neovessels can cause intraplaque hemorrhages, and promote plaque vulnerability and rupture. Plaque rupture leads to activation of coagulation and the formation of a thrombus, which is the main contributor to acute manifestations, morbidity, and mortality in atherosclerotic disease. The extrinsic coagulation activator tissue factor (TF) and its inhibitor tissue factor pathway inhibitor (TFPI) have been detected in atherosclerotic lesions, and are expressed by macrophages, smooth muscle cells, and endothelial cells overlaying the plaques. We aimed to investigate how hypoxic conditions affected the endothelial expression and activity of coagulation factors important in the initiation of coagulation. Hypoxia was induced in primary human umbilical (HUVEC) and aortic (HAEC) endothelial cells with chemicals (DMOG/CoCl2) or 1% oxygen (O2) concentration, and mRNA and protein expressions were measured using qRT-PCR and ELISA, respectively. Microscopy of fluorescence labeled cells was used to visualize cell-associated TFPI. Cell-surface FXa activity was measured using a two-stage chromogenic substrate method. Results showed that hypoxia reduced the TFPI mRNA and protein levels in a dose-dependent manner. The effect was apparent on all the protein pools of TFPI; the secreted, the cell-surface associated, and the intracellularly stored, indicating that the regulation occurred at the transcriptional level. The TF mRNA expression was also affected in a dose-dependent way, where low doses of DMOG and CoCl2 resulted in an increased expression, while high dose of DMOG and 1% O2 reduced the TF mRNA levels. Activated endothelial cells showed a dose-dependent regulation of TF expression following hypoxic stimulation, which is identical to the regulation of the FXa activity in the cells. Our findings indicate that hypoxic conditions can influence the coagulant activity of endothelial cells, which may potentially promote clinical events and thus the severity of atherosclerotic disorders.